In the last decade, the important role of small non-coding RNAs in regulation have been recognized and begun to be studied. Our laboratory, in collaborations with a number of groups, have undertaken two global searches for non-coding RNAs in E. coli, contributing significantly to the more than 80 that are now identified. Using immunoprecipitation and analysis with microarrays, we find that at least one-third of these RNAs bind tightly to the RNA chaperone Hfq. We and others have shown that every RNA that binds tightly to Hfq acts by pairing with target mRNAs, regulating stability and translation of the mRNA, either positively or negatively. Our lab has studied a number of these small RNAs in detail. We have found that expression of each small RNA is regulated by different stress conditions, and that the small RNA plays an important role in adapting to stress. The best-studied of these is RyhB. RyhB transcription is repressed by the Fur iron-dependent repressor, and the small RNA is therefore made in high quantities when intracellular iron is limiting. When it is made, it targets mRNAs that encode iron-binding proteins for degradation. Therefore, this small RNA, which is also found in Vibrio, Salmonella, Klebsiella, and Yersinia, reprograms iron use in the cells and may be an important component of virulence for some pathogens. Two other small RNAs, now called OmrA and OmrB, have been found to regulate a number of outer membrane proteins; these small RNAs are made at high osmolarity are part of the OmpR/EnvZ regulon. Studies on the regulation of RybB, another Hfq-binding RNA, have demonstrated that it is dependent on an alternative sigma factor, Sigma E, for transcription. Sigma E becomes active when misfolded outer membrane proteins accumulate in the periplasm of the cell. RybB autoregulates sigma E, possibly at both the level of synthesis and activity. The demonstration of the activity of RybB suggests that trafficking to the outer membrane may be even more tightly regulated than previously known. These RNAs are characteristic of a growing family of regulatory RNAs that regulate the cell surface, possibly important during infection. Another small RNA, now named SgrS, is made when cells accumulate glucose-6-phosphate or a phosphorylated glucose analog, and down-regulates the mRNA for the . glucose-specific transporter, encoded by ptsG. SgrS induction depends on a novel transcriptional regulator, encoded by the divergent gene, named by us sgrR. The SgrR protein, which is the first studied member of a conserved family of transcriptional regulators (previously mis-annotated), directly binds DNA, negatively autoregulates, and may directly sense the accumulation of sugar phosphate. When either the small RNA or the transcriptional regulator are mutant, cells are unable to recover from glucose-phosphate accumulation. Previous studies had demonstrated the roles of two small RNAs, DsrA and RprA, in positively regulating translation of the stress sigma factor RpoS. More recently, we have shown that RprA also has a number of other mRNA targets, which are negatively regulated; this had been previously shown for DsrA. The new RprA targets expand the likely role of RprA and its regulators, RcsC, RcsD, and RcsB, in controlling biofilm formation by this bacteria. Studies on the mechanism of action of DsrA and RprA suggest that they affect both the stability and translation of rpoS mRNA. RpoS is subject to control at the level of protein turnover as well. RpoS is rapidly degraded during active growth, in a process that requires the energy-dependent ClpXP protease and the adaptor protein RssB, a phosphorylatable protein that presents RpoS to the protease. RpoS becomes stable after various stress or starvation treatments; the mode of stabilization has been a mystery. Recent studies in our labs and others demonstrated significant regulation of degradation in the absence of phosphorylation.